Research On The Expression,Function And Regulation Mechanisms Of Long Non-coding RNA UCA1 In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma (HCC) ranks the fourth most common malignant tumors in our country, and its incidence shows a trend of rising year by year. Due to its high malignancy, quick progress, and poor prognosis, HCC is the second leading cause of cancer-related mortality among the malignant tumors. Therefore, novel diagnostic biomarkers and therapeutic strategies are urgently needed in order to explore the pathogenesis of HCC and improve the clinical diagnosis and treatment for HCC patients. Long non-coding RNAs (IncRNAs) are recently found a class of non-protein-coding RNAs which are closely related to the occurrence and development of the majority malignancies. Recently, studies have showed that IncRNAs may act as competing endogenous RNAs (ceRNAs), which can combine with miRNA response elements (MREs) and inhibit miRNAs functions and activities, subsequently modulating the derepression of miRNA targets at the level of post-transcriptional regulation. LncRNA-urothelial carcinoma associated 1 (UCA1) has firstly been reported in bladder cancer. UCA1 it is highly expressed in bladder cancer, colorectal cancer, breast cancer, etc., and it closely related with the malignant progress of these tumors. Nevertheless, up to now, there is no relevant report about the expression pattern of UCA1 and its underlying mechanism involving in HCC.Objective:To investigate the expression pattern and clinical significance of UCA1 in HCC, and analyze the effects of RNA interference (RNAi) UCA1 on HCC cell proliferation, colony formation, cell cycye, cell invasion and migration, and the growth of transplanted tumor in nude mice. Moreover, to further explore the mechanisms of the UCA1-miRNA-mRNA-pathway regulatory network based on ceRNA mode in the tumorigenesis of HCC.Methods:(1) Screening lncRNA molecules in HCC tissues by lncRNA microarray. (2) Verification the screening lncRNA molecule-UCA1 by qRT-PCR. (3) Analysis the relationship of UCA1 with clinical features and prognosis of HCC patients with Fisher exact test, Kaplan-Meier survival curve, and Cox regression model. (4) Construction the short hairpin RNAi vector targeting UCA1, transfection in HCC cell lines which highly expressed UCA1, and detection the interference efficiency. (5) Detection of cell proliferation by CCK-8 kit, cell cycle by flow cytometry, colony formation by plate method, cell invasion and migration by Transwell assay, and transplanted tumor in nude mice to analyze the biological functions of UCA1. (6) Bioinformatic prediction miRNA which could interact with UCA1 and prediction the target gene to miRNA. (7) qRT-PCR and immunohistochemical analyses the relationship of expression levels among UCA1, miRNA and its target gene in HCC tissues. (8) Verification UCA1 combination with miRNA in RISC by dual-luciferase reporter gene assay and RIP assay. (9) Analysis whether UCA1 could inhibit the biological function of miRNA in HCC. (10) Verification the relationship between miRNA and its target gene by dual-luciferase reporter gene assay. (11) qRT-PCR and Western blotting analyses the effects of UCA1 and miRNA on the mRNA and protein expression of target gene. (12) Western blotting analysis the expression changes of protein and phosphorylation protein in signaling pathways related with target geneResults:(1) By using lncRNA microarray screening and qRT-PCR verification, UCA1 is aberrantly upregulated in HCC tissues than adjacent nontumourous tissues, P<0.001. (2) Clinicopathological analysis showed that UCA1 was significantly correlated with TNM stage and distant metastasis, P<0.001; and high UCA1 expression was significantly associated with poor overall survival rate of HCC patients, P<0.001. Moreover, univariate and multivariate Cox regression analyses showed that UCA1 was an independent prognostic factor for HCC patients. (3) Two RNAi vectors targeting UCA1 were constructed and trandfected into SMMC-7721 and HepG2 cell lines (highly-expressed UCA1). The interference efficiencies were siUCA1#1,81% and 78%; siUCA1#2,54% and 47%; siUCA1#1+siUCA1#2,66% and 60%, respectively. Then, siUCA1#1 was used for further UCA1 functional studies. (4) SiUCA1 could suppress HCC cell proliferation, colony formation, cell migration and invasion and induce GO/Gl cell cycle arrest in vitro. Moreover, siUCAl could inhibit tumor growth in vivo. (5) Bioinformatic prediction showed that miR-216b、miR-665、miR-326、miR-212-5p、miR-338-3p、miR-567 and miR-136-3p had MREs which could be combined with UCA1. The expressions of almost all the miRNAs were not or slightly changed (<1.5-fold) with the exception of miR-216b, miR-216b expression showed a>2-fold increase in siUCA1 transfected HCC cells. (6) The luciferase reporter targeting UCA1 was constructed and co-transfected miR-216b in HCC cells, dual-luciferase reporter gene assay showed that UCA1 and miR-216b were combined with MRE. RIP assay further confirmed that UCA1 and miR-216b were interaction in RISC. (7) MiR-216b expression was dropped in HCC tissues (P<0.01) and an inverse correlation was noted between miR-216b and UCA1 expression levels in HCC tissues, r=-0.6224, P<0.0001. (8) MiR-216b could suppress HCC cell proliferation, colony formation, cell migration and invasion and induce GO/G1 cell cycle arrest and inhibit tumor growth as well. However, UCA1 reverse the inhibitory effect of miR-216b on the growth and metastasis of HCC cells. (9) Bioinformatic prediction showed that FGFR1 was a target gene to miR-216b. Dual-luciferase reporter gene assay further confirmed the prediction. MiR-216b or siUCAl could downregulate the expressions of FGFR1 mRNA and protein in HCC cells. Overexpression of UCA1 could upregulate the expressions of FGFR1 mRNA and protein in HCC cells. However, miR-216b and UCA1 co-overexpression could regain the expressions of FGFR1 mRNA and protein in HCC cells. (10) A significant positive correlation was found between FGFR1 protein or mRNA expression and UCA1 expression in HCC tissues (r=0.7114 and 0.6116, P<0.0001); whereas, a significant negative correlation was found between FGFR1 protein or mRNA expression and miR-216b expression in HCC tissues (r=-0.5040 and-0.7094, P<0.0001). (11) Western blotting analyses showed that UCA1 might facilitate HCC malignant progression partly through FGFR1/ERK, rather than FGFR1/JNK or FGFRl/p38 MAPK, signaling pathway.Conclusion:UCA1 is highly expressed in HCC and is related with TNM stage, metastasis and prognosis of HCC patients. As a potential oncogene, UCA1 can promote HCC cell proliferation, migration and invasion and tumor growth in nude mice. Mechanism research elucidates a novel UCA1-miR-216b-FGFR1-ERK signaling pathway regulatory network, that is UCA1 can act as a ceRNA and combine with miR-216b in RISC, which can reverse the inhibitory effect of miR-216b on the growth and metastasis of HCC cells, resulting in derepression of FGFR1 (miR-216b target gene) expression and activation of FGFR1/ERK signaling pathway, thereby promotion the malignant progression of HCC. Therefore, the findings provide a new clue for understanding the pathogenesis of HCC and provide an intriguing IncRNA-directed approach for the diagnosis and treatment of HCC.